Figure 10. dMi-2 decreases the stability and extent of cohesin chromosome binding.

(A) FRAP recovery curves for EGFP-Smc1 in wild-type salivary gland nuclei, and salivary gland nuclei in which da-GAL4 drives the expression of two UAS-dMi-2⁺ transgenes (EGFP-Smc1 da-GAL4/UAS-dMi-2⁺ 3-3; UAS-dMi-2⁺ 15-1/+) at 25°C. This induced a 1.6-fold average increase in nuclear volume (see text). EGFP-Smc1 was bleached in half the nucleus, and the curves show the decreasing difference in fluorescence intensity (Δ fluorescence) between the bleached and unbleached halves over time as the unbleached EGFP-Smc1 re-equilibrates throughout the nucleus. The curves shown are the average of at least 29 nuclei, and the error bars show the standard error of the means. (B) Curve-fitting distinguishes three cohesin fractions, unbound (determined by the loss of fluorescence in the unbleached half during bleaching, not shown), a weak binding fraction, and a stable binding fraction, presumed to be bound topologically. (C) Both the half-life of the stable binding cohesin (upper right panel) and total fraction of cohesin that binds in the stable mode were decreased by dMi-2 overexpression.